Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome, characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. Patients with DC have a propensity to develop a variety of cancer, in particular of the hematopoietic system and of the upper gastrointestinal tract. The overall aim of our research is to understand the pathophysiology and pathogenesis of DC and the molecular pathways that lead to BMF and cancer susceptibility in patients with this disease. DC is genetically heterogeneous with autosomal dominant, autosomal recessive, and X-linked forms of the disease. The different patterns of inheritance suggest that mutations in different genes may lead to the same phenotype, and that these gene products might function in a common pathway. Patients with the X-linked form of DC have mutations in the dyskerin (DKC1) gene. Dyskerin is a nucleolar protein that associates with small nucleolar RNAs and catalyzes the conversion of specific uridine residues to pseudouridine in 18S and 28S ribosomal RNA. Dyskerin is also a component of the telomerase complex responsible for the maintenance of telomeres, nucleoprotein structures that cap the ends of chromosomes. In the proposed studies we will investigate the biochemical and molecular consequences of dyskerin mutations. We hypothesize that dyskerin is essential in ribosomal RNA processing and in the assembly of an active telomerase complex, and that mutations in dyskerin causing DC may affect ribosomal RNA processing, the telomerase complex, or both, to a variable extent depending on their location in dyskerin. To test this we will study the consequences of a dyskerin null mutation on pseudouridylation, ribosomal RNA processing, ribosome biogenesis, telomerase activity, and the integrity of the telomerase complex. Mouse lines and embryonic stem (ES) cells have been constructed in which a deletion in the dyskerin gene can be induced by the expression of the recombinase Cre. Experiments will be performed in mouse tissues (liver cells, bone marrow cells), and ES cells thus enabling us to study the effects of genetic alteration of dyskerin in normal mammalian cells with a high level of dyskerin and telomerase expression. Next, we will introduce a series of dyskerin mutations into the genome of dyskerin null ES cells and characterize the extent to which these mutations affect the dual function of dyskerin. Finally, we will test whether over-expression of dyskerin, or of telomerase may correct the cellular phenotype caused by DC mutations. Findings are likely to provide important insights into the functional role of dyskerin and dyskerin mutations which will be essential for a better understanding of the pathogenesis and biology of the disease. A better understanding of the disease will provide the framework for the design of novel and more efficient treatment for patients with DC. New insights gained into the importance of ribosome biogenesis and telomere maintenance in human disease might also shed light on the pathogenesis of other bone marrow failure syndromes in which telomere maintenance and ribosome biogenesis is impaired.